This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Background: For established cellulose-based H2-producing co-culture systems, modest H2 production has been achieved in bioreactors with syntrophic microorganisms. However, the metabolic interactions between the organisms are poorly understood. This shortcoming hinders system optimization for improving H2 production efficiency. Methods: We achieved a co-culture system for H2 production based on cellulosic materials with Clostridium cellulolyticum H10 and Rhodopseudomonas palustris CGA584. Dark fermentation by H10 decomposes cellulose into small organic acids, CO2 and H2. CGA584 cannot use cellulose or glucose directly, though it can further degrade fermentation products produced and released in large quantities by H10, yielding additional H2. A stable co-culture was established and the kinetics and stoichiometry of the syntrophic growth were monitored. We have identified several of the organic acids produced and released by H10, but it is important to determine whether other, previously unidentified metabolites are released by H10 and thus become available for uptake and metabolism by CGA584. Therefore, we propose to use AMS to determine the presence or absence of additional metabolites released by H10 that are not detectable using HPLC with UV-Vis and refractive index detection. Results from these experiments will provide the basis for further characterization of the metabolic interaction between H10 and CGA584 in the production of H2 as a biofuel using cellulose as a feedstock. Expected Results and Future Directions: We anticipate that we be able to determine whether previously unidentified metabolites are secreted by H10, which will allow us to develop methods to identify those metabolites and monitor them in future experiments. These experiments will also confirm that metabolites secreted by H10 are the sole carbon source for CGA584. This information, and data from the additional experiments it will enable, will provide a basis for computational modeling of the interaction between H10 and CGA584 in the production of H2 fuel as a result of cellulose degradation. A valid computational model can then be used to predict optimal growth conditions for biofuel production.